Methotrexate (MTX) is a widely used and important agent in the treatment of breast cancer. MTX is often used in combination with other agents in the treatment of breast cancer. MTX is often used in combination with other agents in the treatment of breast cancer. These MTX regimens used in breast cancer treatment include cyclophosphamide, MTX, and 5-fluorouracil (CMF) and cyclophosphamide, MTX, 5-fluorouracil and prednisone (CMFP). Intracellularly, MTX bindings to dihydrofolate reductase (DHFR) at the folate binding site and inhibits the enzyme. DHFR is ultimately required for the production of thymidylate nucleotides which are essential for the synthesis of DNA. Chronic and high dose of MTX is often hampered by the development of MTX resistance in the host cells. MTX resistance is often manifested as a relapse of cancerous growth during treatment or during post-treatment evaluations. MTX resistance may occur via several different molecular mechanisms. These include 1) amplification of the DHFR gene, 2) synthesis of mutant DHFRs, 3) diminished transport of MTX inside cells, 4) decreased ability to synthesize polyglutamates, 5) decreased thymidylate synthase activity. The objective of the proposed research is to use computer-assisted molecular modeling and design methods in conjunction with the available detailed three-dimensional structural knowledge of the folate binding site of human wild type and MTX resistant mutant dihydrofolate reductase (DHFRs) to design new anti-folate analogs which do not required intracellular polyglutamation nor a membrane carrier for transport into host cells. These new agents will also be capable of effectively inhibiting both the wild type and MTX resistant mutant DHFR molecules. This group of new agents will not be susceptible to three of the five known mechanisms of MTX resistance and are expected to represent a significant advancement in the treatment of breast cancer.